Use of thrombin-derived peptides for the therapy of chronic dermal ulcers

ABSTRACT

Disclosed is a method of promoting healing of a chronic dermal skin ulcer, such as a diabetic ulcer, on a subject. The method comprises the step of contacting the chronic dermal skin ulcer with an effective amount of an agonist of the non-proteolytically activated thrombin receptor.

RELATED APPLICATIONS

This application is a continuation of International Application No.PCT/US02/01151, which designated the United States and was filed Jan.16, 2002, published in English, which claims the benefit of U.S.Provisional Application No. 60/308,198, filed Jul. 27, 2001. The entireteachings of the above applications are incorporated herein byreference.

GOVERNMENT SUPPORT

The invention was supported, in whole or in part, by grant 1R01-GM47572and R44-DK53580 from the National Institutes of Health. The Governmenthas certain rights in the invention.

BACKGROUND OF THE INVENTION

Dermal skin ulcers are an example of wounds that are particularlydifficult to treat because they resist healing and consequently oftenbecome chronic wounds. Examples of chronic dermal ulcers include thoseresulting from venous disease (venous stasis ulcers), excessive pressure(decubitus ulcers), arterial ulcers and diabetic ulcers.

Diabetic ulcers are particularly problematic. For example, one in sevenindividuals with diabetes develops dermal ulcers on their extremities,which are susceptible to infection. Treatment of diabetic ulcers isoften prolonged, intensive and costly and treatment failures are common.Current approaches include debridement, frequent changes of wounddressing, specially fitted footwear, oral or intravenous antibiotics,complete bed rest, lengthy hospitalization and surgicalrevascularization. Ulcer-related complications can in some cases requireamputation. Therefore, there is a need for treatments which acceleratethe rate of the healing of chronic dermal skin ulcers in general, and ofdiabetic ulcers, in particular.

SUMMARY OF THE INVENTION

It has now been found that agonists of the non-proteolytically activatedthrombin receptor are effective in accelerating the rate of healing ofdiabetic ulcers. For example, the thrombin peptide derivative TP508,administered topically twice a week at doses of 1.0 μg or 10.0 μgincreased the rate at which diabetic ulcers healed and increased thepercentage of patients who experienced 100% closure of the ulcer. Basedon this discovery, methods of promoting or accelerating healing ofchronic dermal ulcers are disclosed herein.

One embodiment of the present invention is a method of promoting healingof a chronic dermal skin ulcer on a subject. The method comprises thestep of contacting the chronic dermal skin ulcer with an effectiveamount of an agonist of the non-proteolytically activated thrombinreceptor.

The thrombin peptide derivatives used in the methods disclosed hereinare inexpensive to produce and are effective in accelerating the rate atwhich chronic dermal skin ulcers heal and in increasing the likelihoodof complete closure of the ulcer. They also cause few, if any, sideeffects.

DETAILED DESCRIPTION OF THE INVENTION

Dermal skin ulcers refer to lesions on the skin caused by superficialloss of tissue that fail to heal normally due to defects in healingprocesses, vascular insufficiency or pressure. Dermal skin ulcers whichcan be treated by the method of the present invention include decubitusulcers, diabetic ulcers, venous stasis ulcers and arterial ulcers.Decubitus wounds refer to chronic ulcers that result from pressureapplied to areas of the skin for extended periods of time. Wounds ofthis type are often called bedsores or pressure sores. Venous stasisulcers result from the stagnation of blood or other fluids fromdefective veins. Arterial ulcers refer to necrotic skin in the areaaround arteries having poor blood flow.

Applicants have discovered that compounds which stimulate or activatethe non-proteolytically activated thrombin receptor (hereinafter “NPAR”)promote or stimulate healing of chronic dermal skin ulcers. Compoundswhich stimulate NPAR are said to be NPAR agonists. NPAR is ahigh-affinity thrombin receptor present on the surface of most cells.This NPAR component is largely responsible for high-affinity binding ofthrombin, proteolytically inactivated thrombin, and thrombin derivedpeptides to cells. NPAR appears to mediate a number of cellular signalsthat are initiated by thrombin independent of its proteolytic activity.An example of one such signal is the upregulation of annexin V and othermolecules identified by subtractive hybridization (see Sower, et. al.,Experimental Cell Research 247:422 (1999)). NPAR is thereforecharacterized by its high affinity interaction with thrombin at cellsurfaces and its activation by proteolytically inactive derivatives ofthrombin and thrombin derived peptide agonists as described below. NPARactivation can be assayed based on the ability of molecules to stimulatecell proliferation when added to fibroblasts in the presence ofsubmitogenic concentrations of thrombin or molecules that activateprotein kinase C as disclosed in U.S. Pat. Nos. 5,352,664 and 5,500,412.The entire teachings of these patents are incorporated herein byreference. NPAR agonists can be identified by this activation or bytheir ability to compete with ¹²⁵I-thrombin binding to cells.

NPAR agonists include thrombin derivatives described in U.S. Pat. Nos.5,352,664 and 5,500,412. For example, a thrombin peptide derivative cancomprise a thrombin receptor binding domain having the L-amino acidsequence Arg-Gly-Asp-Ala (SEQ ID NO: 7), and a serine esterase conservedsequence. In on embodiment, a peptide derivative of thrombin comprises aserine esterase conserved sequence,Asp-Ala-Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val (SEQ ID NO: 8). Oneexample of an NPAR agonist is a thrombin peptide derivative, i.e., apolypeptide with less than about fifty amino acids, preferably less thanabout thirty-three amino acids and having sufficient homology to thefragment of human thrombin corresponding to prothrombin amino acids508–530(Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val:SEQ ID NO.: 1) that the polypeptide activates NPAR. The thrombin peptidederivatives described herein preferably have between about 14 and 23amino acids, more preferably between about 19 and 23 amino acids,Optionally, the thrombin peptide derivatives described herein can beamidated at the C-terminus and/or acylated at the N-terminus. In oneembodiment, the thrombin peptide derivative being administered to thechronic skin ulcer has the following amino acid sequence:R1-Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val-R2:SEQ ID NO.: 5. R1 is —H or R3-C(O)—; R2 is —OH or —NR4R5; R3 is —H or C1–C6 alkyl group (preferably —CH₃); and R4 and R5 are independently —H,C1–C6 alkyl group or, taken together with the nitrogen atom to whichthey are bonded, are a non-aromatic heterocyclic group such apiperidinyl, morpholinyl, thiomorphinyl or pyrollidinyl (preferably R4and R5 are both —H). Preferably R1 is —H and R2 is —NH₂; or R1 is —H andR2 is —OH. Alternatively, the thrombin peptide derivative beingadministered to the chronic skin ulcer has the amino acid sequence ofSEQ ID NO.: 3:R1-Asp-Asn-Met-Phe-Cys-Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val-Met-Lys-Ser-Pro-Phe-R2.R1 and R2 are as described above. It is understood, however, that zero,one, two or three amino acids at positions 1–9 and 14–23 in the thrombinpeptide derivative can differ from the corresponding amino acid in SEQID NO.: 5. It is also understood that zero, one, two or three aminoacids at positions 1–14 and 19–33 in the thrombin peptide derivative candiffer from the corresponding amino acid in SEQ ID NO.: 3. Preferably,the amino acids in the thrombin peptide derivative which differ from thecorresponding amino acid in SEQ ID NO.: 3 or SEQ ID NO.: 5 areconservative substitutions, and are more preferably highly conservativesubstitutions. Alternatively, an N-terminal truncated fragment of thethrombin peptide derivatives having at least fourteen amino acids or aC-terminal truncated fragment of the thrombin peptide derivative havingat least eighteen amino acids can be contacted with the chronic skinulcer.

A thrombin receptor binding domain is defined as a polypeptide sequencewhich directly binds to the thrombin receptor and/or competitivelyinhibits binding between high-affinity thrombin receptors andalpha-thrombin.

A domain having a serine esterase conserved sequence comprises apolypeptide sequence containing at least 4–12 of the N-terminal aminoacids of the dodecapeptide previously shown to be highly conserved amongserine proteases (Asp-X₁-Cys-X₂-Gly-Asp-Ser-Gly-Gly-Pro-X₃-Val; SEQ IDNO: 9); wherein X₁ is either Ala or Ser; X₂ is either Glu or Gln; and X₃is either Phe, Met, Leu, His, or Val).

A “C-terminal truncated fragment” refers to a fragment remaining afterremoving an amino acid or block of amino acids from the C-terminus. An“N-terminal truncated fragment” refers to a fragment remaining afterremoving an amino acid or block of amino acids from the N-terminus. Itis to be understood that the terms “C-terminal truncated fragment” and“N-terminal truncated fragment” encompass acylation at the N-terminusand/or amidation at the C-terminus, as described above.

A peptide is acylated at the N-terminus when the amine —NH₂ at theN-terminus is derivatized as an acyl group R3-C(O)—NH—, wherein R3 is asdescribed above. Thus, when R1 is R3-C(O)—, the N-terminus is an acylgroup; and when R1 is —H, the N-terminus is an unsubstituted amine.

A peptide is amidated at the C-terminus when the carboxylic acid —COOHat the C-terminus is derivatized as an amide —CONR4R5, wherein R4 and R5are as described above. Thus, when R2 is —OH, the C-terminus is acarboxylic acid; and when R2 is —NR4R5, the C-terminus is amidated.

A preferred thrombin peptide derivative for use in the disclosed methodhas the amino acid sequence of SEQ ID NO.: 2: R1Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Cys-X₁-Gly-Asp-Ser-Gly-Gly-Pro-X₂-Val-R2.R1 and R2 are as described above. Another preferred thrombin peptidederivative for use in the disclosed method has the amino acid sequenceof SEQ ID NO.: 4:R1-Asp-Asn-Met-Phe-Cys-Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Cys-X₁-Gly-Asp-Ser-Gly-Gly-Pro-X₂-Val-Met-Lys-Ser-Pro-Phe-R2.X₁ is Glu or Gln; X₂ is Phe, Met, Leu, His or Val; and R1 and R2 are asdescribed above. Alternatively, N-terminal truncated fragments of thesepreferred thrombin peptide derivatives, the N-terminal truncatedfragments having at least fourteen amino acids or C-terminal truncatedfragments of these preferred thrombin peptide derivative, the C-terminaltruncated fragments having at least eighteen amino acids, can also beused in the disclosed method.

TP508 is an example of a thrombin peptide derivative and has the aminoacid sequence of SEQ ID NO.: 5, wherein R1 is —H and R2 is —NH₂ (SEQ IDNO.: 6). Another example of a thrombin peptide derivative has the aminoacid sequence of SEQ ID NO.: 5, wherein R1 is —H and R2 is —OH (“deamideTP508”). Other examples of thrombin peptide derivatives which can beused in the disclosed method include N-terminal truncated fragments ofTP508 (or deamide TP508), the N-terminal truncated fragments having atleast fourteen amino acids or C-terminal truncated fragments of TP508(or deamide TP508), the C-terminal truncated fragments having at leasteighteen amino acids.

A “conservative substitution” is the replacement of an amino acid withanother amino acid that has the same net electronic charge andapproximately the same size and shape. Amino acids with aliphatic orsubstituted aliphatic amino acid side chains have approximately the samesize when the total number carbon and heteroatoms in their side chainsdiffers by no more than about four. They have approximately the sameshape when the number of branches in the their side chains differs by nomore than one. Amino acids with phenyl or substituted phenyl groups intheir side chains are considered to have about the same size and shape.Listed below are five groups of amino acids. Replacing an amino acid ina polypeptide with another amino acid from the same group results in aconservative substitution:

-   -   Group I: glycine, alanine, valine, leucine, isoleucine, serine,        threonine, cysteine, and non-naturally occurring amino acids        with C1–C4 aliphatic or C1–C4 hydroxyl substituted aliphatic        side chains (straight chained or monobranched).    -   Group II: glutamic acid, aspartic acid and non-naturally        occurring amino acids with carboxylic acid substituted C1–C4        aliphatic side chains (unbranched or one branch point).    -   Group III: lysine, ornithine, arginine and non-naturally        occurring amino acids with amine or guanidino substituted C1–C4        aliphatic side chains (unbranched or one branch point).    -   Group IV: glutamine, asparagine and non-naturally occurring        amino acids with amide substituted C₁–C₄ aliphatic side chains        (unbranched or one branch point).    -   Group V: phenylalanine, phenylglycine, tyrosine and tryptophan.

A “highly conservative substitution” is the replacement of an amino acidwith another amino acid that has the same functional group in the sidechain and nearly the same size and shape. Amino acids with aliphatic orsubstituted aliphatic amino acid side chains have nearly the same sizewhen the total number carbon and heteroatoms in their side chainsdiffers by no more than two. They have nearly the same shape when theyhave the same number of branches in the their side chains. Example ofhighly conservative substitutions include valine for leucine, threoninefor serine, aspartic acid for glutamic acid and phenylglycine forphenylalanine. Examples of substitutions which are not highlyconservative include alanine for valine, alanine for serine and asparticacid for serine.

A “subject” is preferably a human, but can also be an animal in need oftreatment, e.g., companion animals (e.g., dogs, cats, and the like),farm animals (e.g., cows, pigs, horses and the like) and laboratoryanimals (e.g., rats, mice, guinea pigs and the like).

The composition used in the present invention to promote healing ofchronic dermal ulcers can additionally comprise a pharmaceutical carriersuitable for local topical administration in which the thrombin peptidederivative or NPAR agonist is dissolved or suspended. Examples ofpharmaceutically acceptable carriers include, for example, saline,aerosols, commercially available inert gels, or liquids supplementedwith albumin, methyl cellulose or a collagen matrix. Typical of suchformulations are ointments, creams and gels. Ointments are typicallyprepared using an oleaginous base, e.g., containing fixed oils orhydrocarbons, such as white petrolatum or mineral oil, or an absorbentbase, e.g., consisting of an absorbent anhydrous substance orsubstances, for example anhydrous lanolin. Following formation of thebase, the active ingredients are added in the desired concentration.Creams generally comprise an oil phase (internal phase) containingtypically fixed oils, hydrocarbons, and the like, such as waxes,petrolatum, mineral oil, and the like, and an aqueous phase (continuousphase), comprising water and any water-soluble substances, such as addedsalts. The two phases are stabilized by use of an emulsifying agent, forexample, a surface active agent, such as sodium lauryl sulfate;hydrophilic colloids, such as acacia colloidal clays, beegum, and thelike. Upon formation of the emulsion, the active ingredients are addedin the desired concentration. Gels are comprised of a base selected froman oleaginous base, water, or an emulsion-suspension base, as previouslydescribed. To the base is added a gelling agent which forms a matrix inthe base, increasing its viscosity to a semisolid consistency. Examplesof gelling agents are hydroxypropyl cellulose, acrylic acid polymers,and the like. The active ingredients are added to the formulation at thedesired concentration at a point preceding addition of the gelling agentor can be mixed after the gelation process.

The present invention is directed to promoting healing of chronic dermalskin ulcers. A method of treatment “promotes healing” when that thechronic dermal skin ulcer heals more rapidly with the treatment than inthe absence of treatment. Alternatively, a method of treatment“promoting healing” when there is a greater likelihood that the chronicdermal skin ulcer will completely heal than in the absence of thetreatment.

An “effective amount” is the quantity of NPAR agonist or thrombinpeptide derivative which results in greater wound healing and increasedgrowth and proliferation of endothelial cells, keratinocytes andfibroblasts than in the absence of the NPAR agonist or thrombin peptidederivative. Alternatively, an “effective amount” is the quantity of NPARagonist or thrombin peptide derivative which results in a greaterlikelihood that the chronic dermal ulcer will completely heal than inthe absence of the NPAR agonist or thrombin peptide derivative. Theagonist is administered for a sufficient period of time to achieve thedesired therapeutic effect. The amount administered will depend on theamount of dermal growth that is desired, the health, size, weight, ageand sex of the subject, the nature of the chronic dermal skin ulcer(e.g., the type of dermal skin ulcer severity). Typically, between about0.1 μg per day and about 1 mg per day of NPAR agonist or thrombinpeptide derivative (preferably between about 1 μg per day and about 100μg per day) is administered by direct application to the chronic dermalskin ulcer. Generally, enough pharmaceutical carrier or inert solvent isused to cover the wound.

In certain instances where chronic dermal skin ulcers are being treated,it may be advantageous to co-administer one or more additionalpharmacologically active agents to the chronic dermal skin ulcer alongwith a thrombin peptide derivative or NPAR agonist. For example,infection is a threat with any chronic dermal skin ulcer. One aspect ofthe present invention is to co-administer to the chronic dermal skinulcer an antimicrobial, a disinfectant or an antibiotic. Managing painand inflammation are also important aspects of treating chronic dermalskin ulcers. NPAR agonists and thrombin peptide derivatives can also beco-administered to a chronic dermal skin ulcer along with apain-relieving agent such as an analgesic or an anti-inflammatory agent.

Thrombin peptide derivatives can be synthesized by solid phase peptidesynthesis (e.g., BOC or FMOC) method, by solution phase synthesis, or byother suitable techniques including combinations of the foregoingmethods. The BOC and FMOC methods, which are established and widelyused, are described in Merrifield, J. Am. Chem. Soc. 88:2149 (1963);Meienhofer, Hormonal Proteins and Peptides, C. H. Li, Ed., AcademicPress, 1983, pp. 48–267; and Barany and Merrifield, in The Peptides, E.Gross and J. Meienhofer, Eds., Academic Press, New York, 1980, pp.3–285. Methods of solid phase peptide synthesis are described inMerrifield, R. B., Science, 232: 341 (1986); Carpino, L. A. and Han, G.Y., J. Org. Chem., 37: 3404 (1972); and Gauspohl, H. et al., Synthesis,5: 315 (1992)). The teachings of these six articles are incorporatedherein by reference in their entirety.

The invention is illustrated by the following examples which are notintended to be limiting in any way.

Exemplification

Methodology—Study Design

This study was a multi-center, randomized, double blind, three-arm PhaseIIa pilot study evaluating synthetic thrombin peptide TP508 foraccelerating the healing of chronic diabetic ulcers. Patients wererandomized to one of three topical treatment groups: 1 microgram ofTP508 in saline applied twice weekly, 10 micrograms of TP508 in salineapplied twice weekly, or saline placebo applied twice weekly. Allpatients received a regimen of standard diabetic ulcer care consistingof initial sharp debridement, wound cleansing, wound dressing, and woundpressure offloading. Wounds were evaluated twice a week for up to 20weeks or until wound closure, whichever was earlier, Patients wereremoved from the study if they developed a clinical infection or if thewound condition significantly deteriorated. At each wound evaluation(twice weekly), the wound perimeter was traced for determination ofwound area, and the wound was photographed with a digital camera. Bloodchemistry and hematology tests were performed at patient enrollment, andat weeks 5, 10, 15, and 20. A radiographic assessment was conductedevery 5 weeks to study effects on underlying bone composition.

Inclusion/Exclusion Criteria

Males and females ranging in age from 30 to 65 years of age were allowedto participate in the study. Females of childbearing potential had touse an acceptable method of birth control and were urine tested forpregnancy prior to entering the study. To be enrolled in the study, apatient's diabetic pathology had to be clinically documented via bloodglucose and HgbA1C, but other than the diabetic condition, the patienthad to be in reasonably good health. The ulcer to be treated (studyulcer) had to be located below the kneecap, Wagner Grade 1 (wound had topass through the epidermis and into the dermis), Grade 2, or mild Grade3 to the periosteum (without bone or tendon involvement). The studyulcer had to be between 1.0 cm diameter (0.9 cm² area) and 7.0 cmdiameter (about 38.5 cm² area). The study ulcer must also have beenpresent for a minimum of eight (8) weeks and a maximum of two (2) yearswithout healing during that time. The patients' wound oxygen tension(TcPO2) measurement at baseline had to be greater than or equal to 20mmHg as measured by heated oxygen sensors. In addition, patients wererequired to be capable of comprehending and following studyinstructions, complying with the treatment regimen and any prescribedwound pressure offloading, and providing informed consent.

Number of Patients (Planned and Analyzed)

The study was planned for a total enrollment of 60 patients, with 20patients per treatment arm. A total of 60 patients were enrolled andtreated in the study. Of these 60 patients, 12 patients discontinuedfrom the study prematurely. Four patients discontinued due to infectedwounds, two discontinued due to osteomyelitis, one patient discontinuedbecause of amputation, one patient discontinued because of myocardialinfarction, two patients withdrew from the study for non-medicalreasons, one patient due to wound worsening, and one patient took adisallowed medication. None of the causes of study discontinuation wererelated to drug use.

Efficacy (i)

The primary efficacy endpoint was the proportion of patients thatachieve full wound closure. Full wound closure was defined as 100%epithelialization, with no drainage and no infection, as determined byvisual inspection by the clinician. The difference in proportionsbetween the treatment groups was compared. Secondary endpoints includedthe time to 100% closure of the study wound, the time to 80% and 50%wound closure, and the amount of wound closure (as a percentage changefrom baseline wound size) at 3, 5, 10, 15, and 20 weeks.

Efficacy (ii)

Three different patient analysis groups were defined to better study theefficacy endpoints. The Intent-To-Treat (ITT) group included all 60patients receiving study drug and was primarily used for safetyevaluation. The Per-Protocol group included 40 patients that met apredefined set of criteria meant to assure the highest compliance withthe protocol. The Efficacy Group included 46 patients which metstandards that were chosen prior to unblinding to be most relevant toallow an accurate evaluation of wound healing. Primary and secondaryendpoint results are described for each patient group. Similar positivedose response trends for treatment effect in the primary endpoint wereseen in all treatment groups, with the effect most pronounced in thePer-Protocol Group.

Primary Endpoint

The primary efficacy endpoint was the percentage of patients achievingfull wound closure within twenty weeks. The table below summarizes theresults of the primary efficacy endpoint of closure for the threeanalysis populations:

100% Closure Rate Saline (%) 1.0 μg (%) 10.0 μg (%) PP  5/15 (33)  5/11(45)  8/14 (57) ITT 10/21 (48) 11/21 (52) 11/18 (61) EF  6/16 (38)  8/15(53)  9/16 (60) PP—Per Protocol Population ITT—Intent-To-TreatPopulation EF—Efficacy Population

These results show a dose response relationship for 100% closure in allpopulations examined, with 1.0 μg treatments resulting in four tofifteen percent more closure than saline placebo controls, and 10 μgtreatments resulting in thirteen to twenty four percent more closurethan saline placebo controls. Specifically, in the per protocoltreatment group (PP), five of fifteen or 33% healed in the salineplacebo group, five of eleven or 45% healed in the 1 μg treatment group,and eight of fourteen or 57% healed in the 10 μg group. In the efficacy(EF) group selected to include wounds slightly smaller and slightlylarger than those in the stricter per protocol group, this trend wasagain seen with 38% healing in the placebo group, 53% healing in the 1μg group, and 60% healing in the 10 μg group. The difference was againnoted in the intent to treat (ITT) population, although the percentagethat healed in the saline placebo group was larger (48%) because thisgroup included several small and superficial wounds that healed, but didnot meet protocol to be defined as chronic diabetic wounds for thestudy.

These results compare favorably to clinical trials for Regranex®, wheredata compiled from four controlled randomized clinical trials show that83 of 254 or 33% of the vehicle placebo wounds healed by 20 weeks and122 of 285 or 43% of the Regranex®)-treated wounds healed by 20 weeks(see FOI, FDA Clinical Review of BLA-96-1408, OMJ Pharmaceuticals, Dec.9, 1997, page 55, and http/www.fda.gov/cber/products.becamj 121697.htm,updated Mar. 5, 2001 and accessed Jul. 25, 2001; the entire teachings ofthese publications are incorporated herein by reference).

Secondary Endpoints

Secondary endpoints include the time to 100% closure, time to 80%closure, time to 50% closure, and the amount of wound closure as apercentage change from the baseline wound size at 3, 5, 10, 15, and 20weeks. Kaplan-Meier survival analysis techniques were utilized toexamine the time-to-event endpoints.

In the per-protocol population, the median time to 100% closure was 87days for the 10 μg treatment group, versus 122 days for the 1 μgtreatment group. The median time to 100% closure was not reached by 140days in the saline control group. Thus, the healing time in this perprotocol group appears to be shortened in the 10 μg treatment group byat least 53 days relative to placebo controls. The Kaplan-Meier analysisfor time to 100% closure in the per protocol population predicts that by14 weeks there would be a 30% probability of a placebo control woundclosing, about 40% probability of the 1 μg treated wounds closing, andgreater than 60% probability of wounds treated with 10 μg closing.

A similar dose effect was seen in the time to 80% wound closure with themedian time to 80% closure of 32 days for the 10 μg treatment group,versus 47 days for the 1 μg treatment group and 57 days for the salinecontrol group. There was not an obvious dose effect in the time to 50%wound closure with the median time to 50% closure being 21 days in the10 μg group versus 32 days in the 1 μg treatment group and 28 days forthe saline control group.

In the efficacy population, the median time to 100% wound closure was 87days for the 10 μg treatment group versus 116 days for the 1 fμg group.The median time to 100% closure was not reached in the saline controlgroup. For time to 80% wound closure, the median time was 46 days in the10 μg dose group versus 49 days in the 1 μg dose group and 39 days inthe saline control group. For the time to 50% closure, the median timewas 21 days in the 10 μg treatment group versus 29 days and 28 days forthe 1 μg and saline control groups, respectively.

In the intent-to-treat population, the median time to 100% wound closurewas 87 days for the 10 μg treatment group versus 105 days for the 1 μggroup and 102 days for the saline control group. For time to 80% woundclosure, the median time was 31 days in the 10 μg dose group versus 49days in the 1 μg dose group and 29 days in the saline control group. Forthe time to 50% closure, the median time was 17 days in the 10 μgtreatment group versus 29 days and 17.5 days for the 1 μg and salinecontrol groups, respectively.

These results show an increased percentage of ulcer closure for patientstreated with TP508 and indicate median healing times that reflect afaster rate of healing.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

1. A method of treating a chronic skin ulcer in a subject, said methodcomprising administering to the chronic skin ulcer an effective amountof a polypeptide of between 14 to 23 amino acids in length, wherein thepolypeptide comprises a thrombin receptor binding domain of the sequenceArg-Gly-Asp-Ala (SEQ ID NO.: 7) and a serine esterase conservedsequence, wherein the polypeptide is administered to the chronic skinulcer for a duration sufficient to achieve at least 80% closure of thechronic skin ulcer, and wherein the polypeptide is administered to thechronic skin ulcer alone or in combination with an antimicrobial, adisinfectant, an antibiotic, an analgesic or an anti-inflammatory agent.2. The method of claim 1 wherein said serine esterase conserved sequencecomprises Asp-X₁-Cys-X₂-Gly-Asp-Ser-Gly-Gly-Pro-X₃-Val (SEQ ID NO.: 9),wherein X₁ is either Ala or Ser; X₂ is either Glu or Gln; and X₃ iseither Phe, Met, Leu, His, or Val.
 3. The method of claim 2 wherein saidserine esterase conserved sequence comprisesAsp-Ala-Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val (SEQ ID NO.: 8).
 4. Themethod of claim 3 wherein said polypeptide is represented by R1-Ala-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val-R2(SEQ ID NO.: 5), wherein: R1 is —H or R3-C(O)—; R2 is —OH or —NR4R5; R3is —H or a C1–C6 alkyl group; and R4 and R5 are independently —H, aC1–C6 alkyl group or, taken together with the nitrogen atom to whichthey are bonded, a non-aromatic heterocyclic group.
 5. The method ofclaim 4 wherein R1 is —H and R2 is —NH2.
 6. The method of claim 4wherein R1 is —H and R2 is —OH.
 7. A method of treating a chronic skinulcer in a subject, said method comprising administering to the chronicskin ulcer an effective amount of a polypeptide 23 amino acids inlength, wherein the polypeptide comprises the sequenceAla-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val-(SEQ ID NO.: 1), wherein the polypeptide is administered to the chronicskin ulcer for a duration sufficient to achieve at least 80% closure ofthe chronic skin ulcer, and wherein the polypeptide is administered tothe chronic skin ulcer alone or in combination with an antimicrobial, adisinfectant, an antibiotic, an analgesic or an anti-inflammatory agent.8. A method of treating a diabetic ulcer in a subject, said methodcomprising administering to the diabetic ulcer an effective amount ofthe polypeptideAla-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val-NH₂(SEQ ID NO.: 6), wherein the polypeptide is administered to the diabeticulcer for a duration sufficient to achieve at least 80% closure of thediabetic ulcer, and wherein the polypeptide is administered to thediabetic ulcer alone or in combination with an antimicrobial, adisinfectant, an antibiotic, an analgesic or an anti-inflammatory agent.9. A method of treating a decubitus ulcer in a subject, said methodcomprising administering to the decubitus ulcer an effective amount ofthe polypeptideAla-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val-NH₂(SEQ ID NO.: 6), wherein the polypeptide is administered to thedecubitus ulcer for a duration sufficient to achieve at least 80%closure of the decubitus ulcer, and wherein the polypeptide isadministered to the decubitus ulcer alone or in combination with anantimicrobial, a disinfectant, an antibiotic, an analgesic or ananti-inflammatory agent.
 10. A method of treating a venous stasis ulcerin a subject, said method comprising administering to the venous stasisulcer an effective amount of the polypeptideAla-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val-NH₂(SEQ ID NO.: 6), wherein the polypeptide is administered to the venousstasis ulcer for a duration sufficient to achieve at least 80% closureof the venous stasis ulcer, and wherein the polypeptide is administeredto the venous stasis ulcer alone or in combination with anantimicrobial, a disinfectant, an antibiotic, an analgesic or ananti-inflammatory agent.
 11. A method of treating a arterial ulcer in asubject, said method comprising administering to the arterial ulcer aneffective amount of the polypeptideAla-Gly-Tyr-Lys-Pro-Asp-Glu-Gly-Lys-Arg-Gly-Asp-Ala-Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Val-NH2(SEQ ID NO.: 6), wherein the polypeptide is administered to the arterialulcer for a duration sufficient to achieve at least 80% closure of thearterial ulcer, and wherein the polypeptide is administered to thearterial ulcer alone or in combination with an antimicrobial, adisinfectant, an antibiotic, an analgesic or an anti-inflammatory agent.